The present application relates to an optical detector and, more particularly, to an optical detector for use in gene expression analysis, infectious disease testing, gene analysis such as SNP (Single Nucleotide Polymorphism) analysis, protein analysis, cell analysis and other analyses.
Recent years have seen an increasing commercialization of hybridization detection techniques including DNA (deoxyribonucleic acid) chip or DNA microarray. DNA chip is a large number of varied DNA probes packed and immobilized on a substrate surface. Detection of hybridization on the substrate surface of this DNA chip allows for all-inclusive analysis of gene expression and other processes in cells and tissues.
The data obtained from this micro array is verified by means of the realtime PCR (Polymerase Chain Reaction) method. This is a standard approach for quantitative analysis of nucleic acid in trace amounts. The realtime PCR method permits amplification of DNA and other target several-hundreds-of-thousand-fold by repeating the amplification cycle including “thermal denaturation, annealing with a primer and polymerase elongation reaction.” The realtime PCR method is used to monitor in realtime the PCR amplification product obtained as described above for quantitative analysis of nucleic acid in trace amounts. In this method, a dedicated device incorporating a thermal cycler and fluorescence spectrophotometer in a single unit is used to monitor in realtime the PCR amplification product.
In addition to the PCR method, there are other nucleic acid amplification techniques such as the SMAP (SMart Amplification Process), LAMP (Loop-Mediated Isothermal Amplification), NASBA (Nucleic Acid Sequence Based Amplification) and ICAN (Isothermal and Chimeric primer-initiated Amplification of Nucleic acid) methods designed to isothermally amplify DNA. Here, however, a description will be given below of the realtime PCR detection method, a typical amplification method.
First, if only the gene target of interest can be amplified using a highly specific primer, the intercalator method using SYBR (registered trademark) GreenI is employed.
In the intercalator method, an intercalator is used which emits fluorescence when bonded to a double-chain DNA. Fluorescence is emitted by bonding this intercalator to a double-chain DNA produced in the course of PCR reaction and irradiating excitation light onto the intercalator. The amount of PCR amplification product generated is monitored by detecting the intensity of this fluorescence. This intercalator method requires no designing or synthesis of a fluorescent labeling probe specific to the target, making it readily applicable for measurement of a wide variety of targets.
On the other hand, if it is necessary to distinguish between sequences having similar structures or if multiplex detection is required as for typing of SNPs, a probe method is used. The TaqMan (registered trademark) probe method is an example of such a probe method and uses, as a probe, an oligonucleotide having its 5′ end modified with a quencher substance and its 3′ end modified with a fluorescent substance.
The TaqMan probe specifically hybridizes to a template DNA in the annealing step. The same probe does not emit fluorescence when irradiated with excitation light due to the presence of a quencher substance on the probe. In the elongation reaction step, however, the TaqMan probe hybridized to the template DNA is decomposed by a 5′ to 3′ exonuclease activity of the TaqDNA polymerase. As a result, the fluorescent substance is liberated from the probe, eliminating the inhibition by the quencher and causing fluorescence to be emitted. The amount of PCR amplification product generated can be monitored by detecting the intensity of this fluorescence.
A detailed description will be given below of the steps for quantifying gene expression level by the above methods using realtime PCR. First, PCR is performed on a serially diluted standard sample of known concentration as a template to find the threshold cycle (Ct value) required to reach a given amount of amplification product. A standard curve is prepared by plotting this Ct value along the horizontal axis and the initial DNA amount along the vertical axis. Based on this, PCR reaction is performed on a sample of unknown concentration under the same conditions to find the Ct value. Finally, the DNA amount of interest in the sample is measured from this Ct value and standard curve.
As for the techniques related thereto, JP-T-2003-525617 and Japanese Patent Laid-Open No. 2001-136954 (hereinafter referred to as Patent Documents 1 and 2, respectively) disclose, for example, temperature control and other techniques during amplification reaction.